The objective of this proposal is to investigate in detail the mechanisms involved in adenovirus DNA replication. Specially constructed plasmid molecules containing adenovirus origin sequences and inverted repetitious sequences replicate when introduced into cells together with adenovirus DNA or cloned adenovirus genes to provide viral replication proteins in trans. Input plasmid DNA can be distinguished from molecules that have completed one or more rounds of DNA replication by monitoring the state of methylation by cleavage with DpnI, Sau3A, or MboI followed by Southern blot hybridization. This strategy provides a sensitive assay to map the adenovirus origin, to investigate the role of inverted repetitious sequences during replication, and to determine the interplay between the adenovirus origin and trans-acting viral replication proteins. In related work, we have found that adenovirus infection elevates the levels of cellular topoisomerase I. Adenovirus DNA replication requires two cellular factors, one of which can be replaced by purified eukaryotic topoisomerase I. The increase in topoisomerase I activity in infected cells may, therefore, represent an example of viral induction of a cellular factor that is required for viral replication. Preliminary experiments suggest the involvement of E1a in the induction. In order to study the role of viral genes in the induction of topoisomerase I, experiments with E1a mutants are proposed.